Multiplex reverse transcription-PCR screening for EML4-ALK fusion transcripts.
OBJECTIVE
EML4-ALK is a fusion-type protein tyrosine kinase that is generated by inv(2)(p21p23) in the genome of non-small cell lung cancer (NSCLC). To allow sensitive detection of EML4-ALK fusion transcripts, we have now developed a multiplex reverse transcription-PCR (RT-PCR) system that captures all in-frame fusions between the two genes.


METHODS
Primers were designed to detect all possible in-frame fusions of EML4 to exon 20 of ALK, and a single-tube multiplex RT-PCR assay was done with total RNA from 656 solid tumors of the lung (n = 364) and 10 other organs.


RESULTS
From consecutive lung adenocarcinoma cases (n = 253), we identified 11 specimens (4.35%) positive for fusion transcripts, 9 of which were positive for the previously identified variants 1, 2, and 3. The remaining two specimens harbored novel transcript isoforms in which exon 14 (variant 4) or exon 2 (variant 5) of EML4 was connected to exon 20 of ALK. No fusion transcripts were detected for other types of lung cancer (n = 111) or for tumors from 10 other organs (n = 292). Genomic rearrangements responsible for the fusion events in NSCLC cells were confirmed by genomic PCR analysis and fluorescence in situ hybridization. The novel isoforms of EML4-ALK manifested marked oncogenic activity, and they yielded a pattern of cytoplasmic staining with fine granular foci in immunohistochemical analysis of NSCLC specimens.


CONCLUSIONS
These data reinforce the importance of accurate diagnosis of EML4-ALK-positive tumors for the optimization of treatment strategies.
Takeuchi
Kengo
K
Division of Pathology, The Cancer Institute, Tokyo, Japan. kentakeuchi-tky@umin.net
Choi
Young Lim
YL
Soda
Manabu
M
Inamura
Kentaro
K
Togashi
Yuki
Y
Hatano
Satoko
S
Enomoto
Munehiro
M
Takada
Shuji
S
Yamashita
Yoshihiro
Y
Satoh
Yukitoshi
Y
Okumura
Sakae
S
Nakagawa
Ken
K
Ishikawa
Yuichi
Y
Mano
Hiroyuki
H
D000230:Adenocarcinoma / Q000175:diagnosis / Q000235:genetics; D018196:Carcinoma, Adenosquamous / Q000175:diagnosis / Q000235:genetics; D018287:Carcinoma, Large Cell / Q000175:diagnosis / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis / Q000235:genetics*; D002294:Carcinoma, Squamous Cell / Q000175:diagnosis / Q000235:genetics; D002471:Cell Transformation, Neoplastic; D007446:Chromosome Inversion; D017931:DNA Primers / Q000737:chemistry; D005091:Exons / Q000235:genetics*; D015321:Gene Rearrangement; D006801:Humans; D007124:Immunoenzyme Techniques; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; C522504:EML4-ALK fusion protein, human; D015514:Oncogene Proteins, Fusion; D012334:RNA, Neoplasm